Due to increased contamination risks, methods for producing therapeutically active mammalian proteins by isolation and purification from mammalian sources have largely been replaced by recombinant DNA technology. Recombinant DNA technology provides new means for producing large amounts of industrially desirable mammalian proteins and a large choice of hosts and expression systems. When mammalian proteins are the desired target proteins, eukaryotic host systems are preferred in order to obtain glycosylated forms of the desired proteins.
Fungi represent the most effective host system for high volume and low cost production of glycosylated proteins. Even if fungi have been successful for producing high amounts of their native proteins, they have not been equally successful in expressing heterologous proteins, such as therapeutic mammalian proteins.
Fungi are used mainly for large-scale production of homologous industrial enzymes, whereas the focus of research is increasingly in finding alternative expression systems for producing glycosylated heterologous proteins. Because glycosylation patterns and folding processes in plant host systems resemble those in mammalian systems, plant host expression systems are by far the most cost-effective of the available systems. The main interest is on production of pharmaceuticals and industrially important enzymes. The production of interferon, enkephalins, epidermal growth factor and human serum albumin in tobacco, and/or potato are mentioned as examples. The expression levels in transgenic plants, however, have been rather low.
In plant expression systems, foreign genes are generally expressed under strong tissue specific plant promoters in developing plant organs. Typical examples are the seed storage protein promoter or the tuber specific patatin promoter in potato tubers. Alternatively, cell or organ cultures and algal cultures are used as well as even more sophisticated systems including plant virus based systems, with the desired gene coupled inside the virus genome and expressed concurrently with viral proteins. The most advanced systems rely on inducible expression and secretion of recombinant protein to the medium.
The U.S. Pat. No. 5,693,506 and U.S. Pat. No. 5,994,628 disclose a production system for producing foreign proteins in germinating monocotyledonous seeds. Recombinant proteins are expressed under a strong amylase promoter in a cell culture or in germinating seed and the protein is secreted into the growth medium or extracted from malt. In a monocotyledonous seed, the storage reserves mainly consist of the starchy endosperm. The protein stores in monocotyledonous are scarce as compared to those in dicotyledonous seeds. In addition, even if the amylase expression is high in specific cells, only a small number of cells in the seed express amylase. These cells are restricted to the scutellum of the embryo and the aleurone layer of the endosperm. There is a great demand to provide more cost effective systems, which would take advantage of the storage reserves of proteins and oils in dicotyledonous seeds. The U.S. Pat. No. 5,543,576 and U.S. Pat. No. 5,714,474 disclose a method in which transgenic seeds are added without any pregermination and in ground form into feed mixtures as additional enzyme sources.
The U.S. Pat. No. 5,670,349 discloses the use of wound inducible HMGR/HMG2 promoters for expressing recombinant proteins in fresh tobacco leaves harvested from the field. Wounding of fresh or stored plant material by excision or crushing triggers a rapid increase in expression. However, the protein content in tobacco leaf is small as compared to that obtainable from a dicotyledonous seed. Furthermore, the storability of fresh leaves is not comparable to that of dry seed in respect of time, space and/or storage costs. The use of fresh plant material, such as leaves harvested from the field is also a major source of microbial contamination, which is a serious problem in fermentation technology.
The patent applications WO 94/11519 and WO 97/32986 disclose methods and plants for producing degradation and conversion products in plants by the aid of a malting process. In said methods, it is suggested that the enzymes are active in glyoxysomes, which catalyze the breakdown of fatty acids into acetyl-CoA. This acetyl-CoA which normally is used to make organic acids that can be exported from the glyoxysomes and used in other metabolic pathways, such as respiration and sucrose synthesis should be replaced with gene encoding enzymes in a pathway leading to polyhydroxyalkanoates useful for the production of biodegradable thermoplastics.
Even if malting process is known and has been used especially for preparing monocotyledonous plants, it is restricted to processing starch. Even if the methods disclosed in the patents WO 94/11519 and WO 97/32986 suggest the use of the storage reserves in dicotyledonous, these methods are still restricted to the use of enzymes and pathways that lead sucrose and energy production and to use of these products from the respiratory pathways for resynthesis.
Even if malting is known and methods for using the respiratory pathways in plants for production of polyhydroxyalkanoates, the use of the storage reserves in dicotyledonous plants for producing proteinaceous products, such as structural proteins and enzymes has not been solved.
There is a great demand for proteinaceous products and because of that it is the main objective of the present invention to solve the problem of converting the storage reserves in dicotyledonous plants into different proteinaceous products, which can be further applied for production of desired products.
The main objective of the present invention is to provide a new, more feasible, cost-effective, environmentally friendly process and production system for producing gene products, especially proteinaceous gene products in the cotyledons of transgenic dicotyledonous seeds.
Another objective of the present invention is to provide a production system for contained use, in which the gene product can be produced in confined conditions and not in the field. This allows the present process to be carried out under almost contaminant-free conditions.
The objectives of the present invention are achieved by harnessing the regulatory sequences of transient proteins accumulating during the initiation of germination for the production of desired gene products.
The characteristics of the present invention are as set out in the claims.